Conventionally, DNA microarray targeting mRNA is widely used as a means of measuring the gene expression levels in cells. In the 21st century, it is reported that miRNA, Filch is a short-chain and a non-coding RNA, controls gene expression in vivo, and relationships between abnormal expression of miRNA and a variety of diseases such as the cancer have been elucidated. Based on these findings, development race of the DNA microarray targeting miRNA takes place.
Furthermore, the possibility of diagnosis of cancer in which blood test is simply conducted was shown since Mitchell et al, Proc. Nat, Acad. Sci. vol. 105, pp 10513-10518, 2008, showed that miRNA is circulating in the blood in 2008. Accordingly, it is expected that the market of DNA microarray targeting miRNA will rapidly expand in the near future.
On the other hand, quantitative PCR (qRT-PCR) method is widely put to practical use as a technique which quantifies miRNA by using a reverse transcription reaction to cDNA. The quantitative PCR method is a method which converts miRNA to cDNA by using a reverse transcriptase, and amplifies the cDNA, to estimate the amount of miRNA used as a template. The quantitative PCR method has a problem such that parallel processing for multiple samples and various miRNAs is difficult, since it is a method for measuring the amount of cDNA amplified to a certain amount by using PCR reaction, while quantitative capability is higher than that of DNA microarray.
The existing DNA microarrays targeting miRNA (hereinafter referred as miRNA targeting DNA microarrays) are obtained by arranging nucleic acid probes having gene sequences complementarily hybridizing to the objective miRNA a transparent substrate.
As the quantification method of miRNA using miRNA targeting DNA microarrays, for example, the following methods are exemplified. First, after extracting miRNA from a biological sample, fluorescently miRNA, then the labeled miRNA is added to the miRNA targeting DNA microarray to hybridize to the nucleic acid probe on the substrate. Then, after washing miRNA that is nonspecifically adsorbed to the substrate, the amount of miRNA is estimated based on the fluorescence intensity.
When preparing miRNA from a biological sample, total RNA is extracted from the biological sample and purified, then the total RNA including miRNA is fluorescently labeled, then the fluorescently labeled total RNA including fluorescently labeled miRNA is contacted with miRNA targeting DNA microarrays.
However, miRNA is susceptible to the effect of adsorption or decomposition during pipetting in the case such pre-treatment step is complex, because the amount of miRNA in vivo, especially in blood, is 0.01% by mass among total RNA and very small. Furthermore, there is also a problem such that variations in fluorescent labeling index of each measurement arise to lower the reproducibility of the measurement results.
In addition, it is desired that all of the pretreatment steps will be fully automated to be able to be performed on the chip by using μ-TAS (Micro-Total Analysis Systems) in the future, since such hand working pretreatment is affected by the technology differences of scientists or clinical laboratory technicians. Here, the μ-TAS means the microfluidic device analyzing biological molecules on a single chip provided with a small flow path, reaction chamber and mixing chamber on the chip by using the MEMS (Micro Electro Mechanical Systems) technology. However, the fluorescently labeling method becomes a bottleneck as shown below, integration into full automation has not progressed.
The main methods of fluorescently labeling miRNA include a method of fluorescently labeling the base portion of miRNA directly, and a method of adding a fluorescently labeled nucleotide to the 3′ end of miRNA using an enzyme such as T4 DNA ligase.
However, since all of nucleic acids are fluorescently labeled non-specifically by these methods, it is necessary to remove the unreacted fluorescent reagent and the like from the fluorescently pre-labeled target miRNA prior to hybridization to the nucleic acid probe on the substrate. Gel filtration chromatography is generally used to separate these, and it is necessary to accurately separate short miRNA whose length is about 22 bases from the unreacted fluorescent reagent. For example, in the case of separating by using the μ-TAS, a step of migrating a biological sample in an area filled with resin for a long distance is required, and it is extremely difficult to carry out the step in a chip with a limited space. Even if it is possible, in order to repeat the experiment continuously, it is required to flush the unreacted fluorescence reagent by washing for a long time, and it is not practical.
For the above problem, a sandwich-type microarray method which can detect miRNA without the separation process of the unreacted fluorescent dye has been devised in WO2008/052774.
As a first method of the sandwich-type microarray method, concretely, the following steps are exemplified. First, by dual partitioning nucleic acid probes having a sequence complementary to each miRNA 103 which comprises first portion 100 and second portion 101, capture probe 104 (Capture probe) and detection probe 105 (Detect probe) are generated. Then, a microarray is fabricated by arranging the capture probe 104 group having a sequence complementary to the first portion 100 of each miRNA 103 on the substrate 106 (see FIG. 5).
Then, after contacting each miRNA 103 with the fabricated microarray substrates (substrate 106), the tripartite of miRNA 103, capture probe 104 and detection probe 105 are hybridized by contacting a solution containing the detection probe 105 group having a sequence complementary to the second portion 101 of each miRNA 103 with the microarray substrate (substrate 106). It is not necessary to separate the unreacted detection probe 104 by such as chromatography since the detection probe 105 recognizes and binds to the second portion 101 of miRNA 103, and non-specific binding to the capture probe 104 does not occur.
However, in the first method described in WO2008/052774, each sequence complementary to mRNA 103 in one probe becomes about 10 bases since the nucleic acid probe having a sequence complementary to miRNA 103 is divided into two portions. As a result, the affinity of miRNA 103 and the nucleic acid probe is reduced, and it is difficult to accurately quantify miRNA 103 which exists in blood in only trace amounts. In addition, pre-miRNA (precursor of miRNA) of about 70 bases is contained in the biological sample. There is a fundamental problem in the first method of WO2008/052774, it is impossible to accurately quantify only miRNA having a gene expression control function, because pre-miRNA contains a sequence of a miRNA of about 22 bases, and the first method of WO2008/052774 cannot distinguish pre-miRNA and miRNA.
As a solution to this problem, in the second method described in WO2008/052774, further, a method for covalently bonding miRNA 103 and the nucleic acid probe using ligase has been proposed (see FIG. 6). However in the first method shown in FIG. 5, when ligase is used, there is a risk such that the same molecule miRNA 103 is detected more than once, since the capture probe 104 is covalently bonded to the detection probe 105 which hybridized with miRNA 103 then miRNA 103 is dissociated and binds to the other capture probe 104.
On the other hand, in the second method shown in FIG. 6, dissociation of miRNA 103 from the substrate 106 is prevented by further using two kinds of the bridging probes 107, 108 (c-bridge107, d-bridge108) thereby covalently bonding the capture probe 104, miRNA 103 and the detection probe 109 by ligation.